Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.     

Regulation and intracellular localization of the epithelial isoforms of the Na+/H+ exchangers NHE2 and NHE3

The Na+/H+ exchangers (NHE) are a ubiquitous family of membrane proteins that catalyze the counter-transport of extracellular Na+ for intracellular H+ and are important for intracellular pH and cell volume regulation. The major epithelial isoforms, NHE2 and NHE3, are thought to have more specialized roles in regulating Na+ and water absorption and are differentially expressed in epithelial tissues. NHE2 and NHE3 not only differ with respect to their response to various endogenous and exogenous factors but exhibit different intracellular localization as well. NHE2 is primarily located at the plasma membrane, whereas NHE3 is mostly sequestered in an intracellular compartment corresponding to the recycling endosome. Furthermore, NHE3 is localized to the apical pole, whereas polar localization of NHE2 has been controversial. The author has recently localized NHE2 to the apical membrane of a renal epithelial cell line and identified a 45-residue-long region of the cytosolic domain (corresponding to residues 731-777 of the rat NHE2) to be critical for apical targeting. Although SH3 domains of various proteins were found to bind to this and a more carboxy-terminal proline-rich region in vitro, the functional significance of these interactions appears inconsequential. Deletion of both proline-rich regions did not affect Na+/H+ exchange nor its response to hypertonicity and metabolic depletion. However, loss of residues 731-777, which bound specifically in vitro to the SH3 domain of the cytoskeletal protein, alpha-spectrin, mistargets NHE2 to the basolateral surface.     

Na(+)-dependent transporters mediate HCO(3)(-) salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.     

Mineralocorticoids and acidosis regulate H+/HCO3- transport of intercalated cells.

The effects of acidosis and mineralocorticoids on cellular H+/HCO3- transport mechanisms were examined in intercalated cells of the outer stripe of outer medullary collecting duct (OMCDo) from rabbit. Intracellular pH (pHi) of intercalated cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi recovered from an acid load at 2.8 +/- 0.5 x 10(-3) pHU/s in the absence of ambient Na+. This pHi recovery rate was similar in chronic acidosis induced by NH4Cl loading, but it was enhanced (+111%) by treatment with deoxycorticosterone acetate (DOCA). In a DOCA-treated group, luminal 10 microM SCH28080 and 0.1 mM omeprazole, H(+)-K(+)-ATPase inhibitors, did not change the pHi recovery rate, while luminal 0.5 mM N-ethylmaleimide blocked the rate by 68%. DOCA, but not acidosis, increased (approximately 40%) initial pHi response to bath HCO3- or Cl- reduction in Na(+)-free condition. After an acid load in the absence of Na+ and HCO3-, pHi response to basolateral Na+ addition was stimulated (+66%) by acidosis, but not by DOCA. Our results suggest that (a) mineralocorticoids stimulate H+/HCO3- transport mechanisms involved in transepithelial H+ secretion, i.e., a luminal NEM-sensitive H+ pump and basolateral Na(+)-independent Cl(-)-HCO3- exchange; and (b) acidosis enhances the activity of basolateral Na(+)-H+ exchange that may be responsible for pHi regulation.     

Mineralocorticoid modulation of apical and basolateral membrane H+/OH-/HCO3- transport processes in the rabbit inner stripe of outer medullary collecting duct.

To examine the mechanism by which mineralocorticoids regulate HCO3- absorption in the rabbit inner stripe of the outer medullary collecting duct, we microfluorometrically measured intracellular pH (pHi) in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) assaying the apical and basolateral membrane H+/OH-/HCO3- transport processes in three groups of animals: those receiving chronic in vivo DOCA treatment (5 mg/kg per d x 2 wk); those with surgical adrenalectomy (ADX, [chronic x 2 wk]) on glucocorticoid replacement; and controls. Baseline pHi was not different in the three groups. Cellular volume (vol/mm) was increased 38% in DOCA tubules versus controls, but unchanged in ADX tubules versus controls. Buffer capacities (BT) were not different in the three groups. Apical membrane H+ pump activity, assayed as the Na(+)-independent pHi recovery from an acid load (NH3/NH4+ prepulse) and expressed as JH (dpHi/dt.vol/mm.BT) was increased 76% in DOCA tubules versus controls, and decreased 56% in ADX tubules versus controls. Basolateral membrane Cl-/HCO3- exchange activity assayed as the pHi response to basolateral Cl- addition was increased 73% in DOCA tubules versus controls, and decreased 44% in ADX tubules versus controls. When examined as a function of varying [Cl-], the Vmax of Cl-/HCO3- exchange activity was significantly increased in DOCA tubules (control, 72.7 +/- 15.7 pmol.mm-1.min-1 vs DOCA, 132.3 +/- 22.5 pmol.mm-1.min-1, P less than 0.02), while the K1/2 for Cl- was unchanged. Basolateral membrane Na+/H+ antiporter activity assayed as the Na(+)-dependent pHi recovery from an acid load was not changed in chronic DOCA tubules versus controls. In conclusion, the apical membrane H+ pump and basolateral membrane Cl-/HCO3- exchanger of the rabbit OMCDi are regulated in parallel without chronic alterations in pHi under the conditions of mineralocorticoid excess and deficiency. The parallel changes in these transporters accounts for the alterations in OMCDi HCO3- absorption seen under these conditions.     

Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice

To understand the role of Na(+)/H(+) exchanger 1 (NHE1) in intracellular pH (pH(i)) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH(i), using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO(2)/HCO(3)(-), steady-state pH(i) was higher in WT neurons than in mutant neurons. Using the NH(4)Cl prepulse technique, we also show that H(+) flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na(+) from the extracellular solution or by using 100 microM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO(2)/HCO(3)(-), the difference in H(+) flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 microM/s between them at pH 6.6. H(+) flux in CO(2)/HCO(3)(-) was responsive to diisothiocyanato-stilbene-2, 2'-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH(i) and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO(3)(-), indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO(3)(-)-dependent and -independent transporters in neurons.     

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.     

Altered Na+/H+ exchanger isoform 1 activity in immortalized lymphoblasts from women with pre-eclampsia: evidence for an intermediate phenotype

Previous studies have demonstrated a raised Na(+) content in leucocytes isolated from women with pre-eclampsia. Increased Na(+)/H(+) exchanger activity is one membrane transport abnormality that may contribute to this phenomenon and may be implicated in the abnormal volume homoeostasis and hypertension associated with the disease. Increased Na(+)/H(+) exchanger activity has been documented in nucleated white blood cells from both pre-eclamptic and post-partum pre-eclamptic women, and may suggest the importance of genetic influences on exchanger activity. In the present study, we used lymphoblasts from women with pre-eclampsia and from age- and gestation-matched normotensive pregnant controls to determine Na(+)/H(+) exchanger activity and intracellular resting pH using fluorimetry and the pH-sensitive dye BCECF-AM [bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester]. Determination of Na(+)/H(+) exchanger protein abundance was performed by Western blotting. Intracellular pH was not significantly different in cells from pre-eclamptic women compared with those from normotensive controls. Na(+)/H(+) exchanger activity was measured when the intracellular pH was clamped at 6.0, and was found to be significantly higher in cells from pre-eclamptic women (20.77+/-0.92 mmol x min(-1) x l(-1)) compared with those from normotensive controls (15.22+/-0.92 mmol x min(-1) x l(-1); P =0.001). Na(+)/H(+) exchanger protein abundance was established to be similar in the two subject groups, suggesting that the turnover number for the Na(+)/H(+) exchanger is increased in the women with pre-eclampsia. These changes in Na(+)/H(+) exchanger activity indicate the importance of genetic factors in determining this particular phenotype, since in this cell culture model of pre-eclampsia it is likely that environmental or hormonal influences present in vivo would have declined. Overactivity of the Na(+)/H(+) exchanger may contribute to the raised intracellular Na(+) concentration reported previously in white blood cells from women with pre-eclampsia.     

Impairment of sodium pump and Na/H exchanger in erythrocytes from non-insulin dependent diabetes mellitus patients: effect of tea catechins

BACKGROUND: Tea catechins (EGCG, EGC, ECG and EC) possess many important biological properties. We evaluated the effect of tea catechins on erythrocyte membrane Na(+)/K(+)-ATPase and sodium/hydrogen exchanger (NHE) activity in normal (control) and NIDDM subjects. METHODS: Erythrocyte membrane Na(+)/K(+)-ATPase and NHE activity were determined in normal and non-insulin dependent diabetes mellitus (NIDDM) patients. In vitro effect of tea catechins was studied by incubating membrane/intact erythrocytes in assay medium prior to Na(+)/K(+)-ATPase/NHE activity determination. RESULTS: A 24.2% decrease in the activity of Na(+)/K(+)-ATPase (p<0.001) and 39.37% increase in activity of NHE (p<0.02) were observed in NIDDM subjects compared to normal. Tea catechins inhibited the activity of Na(+)/K(+)-ATPase and NHE in both normal and NIDDM erythrocytes, the effect was concentration-dependent. The inhibitory effect of EGCG and ECG at micromolar concentrations was greater compared to EGC and EC on Na(+)/K(+)-ATPase. On NHE the inhibition of tea catechins was in the order: EC>EGC>ECG>EGCG at concentrations up to 10 micromol/l. CONCLUSIONS: This data may help to explain the anti-carcinogenic and cardioprotective effects of tea catechins. The effect of tea catechins on Na(+)/K(+)-ATPase and NHE may be explained due to a direct effect of these compounds on plasma membrane leading to a change in membrane fluidity.     

Inhibition of Rho-kinase reduces renal Na-H exchanger activity and causes natriuresis in rat

Rho-kinase regulates the actin cytoskeleton and therefore modulates transport. The role of Rho-kinase in Na-H exchanger (NHE) activity of rat proximal convoluted tubules (PCTs) was investigated using (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), a specific inhibitor of Rho-kinase. In spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, apical and basolateral NHE activities were determined by measuring cell pH recovery following luminal NH4+ prepulse and basolateral sodium removal, respectively. Apical NHE activity was greater in 8 to 9 week old hypertensive SHR compared with WKY. Although Y-27632 suppressed pH(i) recovery in both strains, sensitivity was 50-fold higher in adult SHR. Y-27632 suppressed basolateral NHE in both strains with similar sensitivity. Apical NHE activity was not greater in 5-week-old, not yet hypertensive, SHR rats compared with WKY. In clearance studies, Na excretion was less in SHR than in WKY rats. Y-27632 increased Na excretion and fractional excretion Na in both strains but more so in SHR. (22)Na uptake of the brush border membrane vesicle taken from Y-27632-treated rats decreased more than that from vehicle-treated animals in both adult SHR and WKY. We conclude that apical NHE activity is increased in SHR PCT compared with controls and that inhibition of Rho-kinase reduces PCT NHE activities and causes natriuresis.     

Basolateral membrane Na+/H+ antiport, Na+/base cotransport, and Na+-independent Cl-/base exchange in the rabbit S3 proximal tubule.

The basolateral membrane Na+ and Cl(-)-dependent acid-base transport processes were studied in the isolated perfused rabbit S3 proximal straight tubule. Intracellular pH (pHi) was measured with 2'7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microfluorometer coupled to the tubule perfusion apparatus. Reduction of basolateral HCO3- from 25 to 5 mM caused pHi to decrease at a rate of 0.81 pH/min. Approximately 50% of this rate was Na+-dependent, 30% Cl(-)-dependent and 20% Na+ and Cl(-)-independent. Two basolateral Na+-dependent acid base transport pathways were detected: (a) an amiloride-sensitive Na+/H+ antiporter and (b) a stilbene-sensitive Na+/base cotransporter. No evidence was found for a Na+-dependent Cl-/base exchanger. The Cl(-)-dependent component of basolateral base efflux was mediated by a stilbene-sensitive Na+-independent Cl-/base exchange pathway. The results suggest that the acid base transport pathways of the basolateral membrane of the S3 proximal tubule differ from more proximal nephron segments.     

Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule

The proximal convoluted tubule (PCT) reabsorbs most of the filtered bicarbonate. Proton secretion is believed to be mediated predominantly by an apical membrane Na(+)/H(+) exchanger (NHE). Several NHE isoforms have been cloned, but only NHE3 and NHE2 are known to be present on the apical membrane of the PCT. Here we examined apical membrane PCT sodium-dependent proton secretion of wild-type (NHE3(+/+)/NHE2(+/+)), NHE3(-/-), NHE2(-/-), and double-knockout NHE3(-/-)/NHE2(-/-) mice to determine their relative contribution to luminal proton secretion. NHE2(-/-) and wild-type mice had comparable rates of sodium-dependent proton secretion. Sodium-dependent proton secretion in NHE3(-/-) mice was approximately 50% that of wild-type mice. The residual sodium-dependent proton secretion was inhibited by 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Luminal sodium-dependent proton secretion was the same in NHE3(-/-)/NHE2(-/-) as in NHE3(-/-) mice. These data point to a previously unrecognized Na(+)-dependent EIPA-sensitive proton secretory mechanism in the proximal tubule that may play an important role in acid-base homeostasis.     

Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.     

Gallbladder Na+/H+ exchange activity is up-regulated prior to cholesterol crystal formation

BACKGROUND: Gallbladder Na+ and H2O absorption are increased prior to gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) isoforms NHE2 and NHE3 are involved in gallbladder Na+ transport in prairie dogs. We examined whether increased gallbladder Na+ absorption observed during early gallstone formation is the result of NHE up-regulation. MATERIALS AND METHODS: Native gallbladder and primary cultures of gallbladder epithelial cells (GBECs) harvested from prairie dogs fed nonlithogenic (CON) or 1.2% cholesterol diet for varying lengths of time to induce cholesterol-saturated bile (PreCRYS), cholesterol crystals (CRYS), or gallstones (GS) were used. NHE activity was assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under H+ gradient in primary GBECs. HOE-694 was used to determine NHE2 and NHE3 contributions. NHE protein and mRNA expression were examined by Western and Northern blots, respectively. RESULTS: Gallbladder total NHE activity was 25.1 +/- 1.3 nmol mg protein(-1) min(-1) in the control and increased during gallstone formation peaking at the PreCRYS stage (98.4 +/- 3.9 nmol mg protein(-1) min(-1)). There was a shift in NHE activity from NHE2 to NHE3 as the animals progressed from no stones through the PreCRYS and CRYS stages to gallstones. The increase in NHE activity was partly caused by an increased Vmax without any change in K(Na)m. Both NHE2 and NHE3 protein increased moderately during the PreCRYS stage without increases in mRNA expression. CONCLUSIONS: Increased gallbladder Na+ absorption observed prior to crystal formation is in part caused by an increase NHE activity which is not fully accounted for by an increase in NHE proteins and mRNA levels but may be explained by enhanced localization in the membranes and/or altered regulation of NHE.     

Na+/Ca2+ exchange inhibitors modulate thapsigargin-induced Ca2+ and Na+ influx in human lymphocytes

Thapsigargin has been shown the elevate intracellular Na(+) concentration in human lymphocytes, but mechanisms underlying thapsigargin-induced Na(+) entry are little understood. In the present study we investigated thapsigargin-induced changes in cytosolic free Na(+) and Ca(2+) concentration in human lymphocytes after inhibition of the Na(+)/Ca(2+) exchange with two structurally unrelated compounds, dimethylthiourea ad bepridil. The intracellular Na(+) increase induced by 5 microM thapsigargin was significantly enhanced in the presence of 5 mM dimethylthiourea or 40 microM bepridil. In contrast, both compounds significantly decreased the thapsigargin-induced intracellular Ca(2+) elevation. No effect of dimethylthiourea or bepridil on thapsigargin-induced Ca(2+) influx was observed in the absence of extracellular Na(+). These observations are consistent with the hypothesis that thapsigargin stimulates Na(+)/Ca(2+ )exchange in human lymphocytes. However, Na(+)/Ca(2+) exchange does not mediate Na(+) influx in human lymphocytes.     

The mechanism of decreased intestinal sodium and water absorption after acute volume expansion in the rat

Studies were performed in rat small intestine in vivo to determine the effect of saline infusion on intestinal transport of Na(+) and H(2)O. Saline infusion decreased net Na(+) flux (J(n) (Na)) from 12.7 +/-0.8 to 6.4 +/-1.5 muEq/hr per cm in the jejunum when the intestinal perfusate contained both Na(+) and glucose. A similar fall in J(n) (Na) occurred in ileum. When mannitol was substituted for glucose in the perfusate, control absorption decreased 29% in jejunum and 18% in ileum, but saline infusion still caused a decrease in J(n) (Na) quantitatively similar to that seen when glucose was present. When choline was substituted for Na(+) in the perfusate, there was net movement of Na(+) from blood to lumen during control and this net secretion was increased further after saline infusion. These observations suggest that saline infusion has a similar effect to decrease intestinal J(n) (Na) under three widely different conditions of basal sodium transport. Permeability of intestinal mucosa to inulin was very low under basal conditions but increased fivefold after saline infusion, and the unidirectional flux of Na(+) from blood to lumen doubled. This increase in unidirectional flux of Na(+) was greater than the observed decrease in J(n) (Na).Thus, saline infusion decreased net absorption of Na(+) and H(2)O from small intestine through mechanisms which did not appear to be dependent upon the rate of Na(+) flux from lumen to blood, and in association with an increased flux of inulin and Na(+) into the intestinal lumen. The data suggest that the effect of saline infusion to decrease net absorption from the intestine could be due either to an increase in passive permeability of the epithelium which could disrupt solute gradients within the membrane or to an increase in flow of solution into the intestinal lumen.     

Na+ gradient-dependent p-aminohippurate (PAH) transport in rat basolateral membrane vesicles

The relationship between the transmembrane Na+ gradient and p-aminohippurate (PAH) transport was examined in isolated rat basolateral membrane vesicles. A 100 mM Na+ gradient (o leads to i) accelerated the influx of 50 microM [3H]PAH whereas similar gradients of choline+, K+, or Li+ did not. The sodium effect was not due to a diffusion potential. The Na+ gradient (o leads to i) decreased the apparent Michaelis constant for PAH from 0.167 +/- 0.016 to 0.054 +/- 0.016 mM and increased the maximum flux rate from 116.00 +/- 13.50 to 427.34 +/- 98.96 pmol/mg/min. An "overshoot" of [3H]PAH influx (159 +/- 4% of the equilibrium value) could be demonstrated only in the presence of a Na+ gradient (o leads to i) plus an opposing gradient of unlabeled PAH (i leads to o). These results suggest that PAH transport and the Na+ gradient are functionally related. A model for cellular uptake of PAH by a Na+ gradient-dependent anion exchange mechanism is presented.     

T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase

Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na(+)/K(+)-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1(-/-)), but not IFN-gamma knockout mice. Anti-CD3 mAb decreased mucosal Na(+)/K(+)-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1(-/-) mice. Neither alpha nor beta subunits of Na(+)/K(+)-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3-treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na(+) absorption, Na(+)-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR(-/-) mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na(+)/K(+)-ATPase activity leading to decreased intestinal Na(+) and water absorption.     

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.     

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.